Anthelmintic bioconversion products

ABSTRACT

There are disclosed new compound with significant antiparasitic activity which are prepared by fermenting the compounds paraherquamide or dihydroparaherquamide in a novel microorganism identified as Cunninghamella blakesleeana MF-4415. The compounds are best realized in the following structural formulae: ##STR1## wherein A is either a single or a double bond. The paraherquamide compound is modified by the microorganism by ring hydroxylation or ketonization. The compounds are significant antiparasitic and anthelmintic agents and compositions for that use are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of copending application Ser.No. 07/602,827 filed Oct. 24, 1990 now abanadoned which in turn is acontinuation of my application Ser. No. 07/330,522 filed Mar. 30, 1989,now abandoned.

BACKGROUND OF THE INVENTION

In Yamazaki et al Tetrahedron Letters 22 135-136 (1981), there is adisclosed paraherquamide which is isolated as a fungal metabolite. Nouses for paraherquamide are disclosed in Yamazaki et al. Also disclosedis the synthetic derivative dihydroparaherquamide produced by thecatalytic reduction of paraherquamide. Paraherquamide anddihydroparaherquamide are best described in the following structuralformula: ##STR2##

SUMMARY OF THE INVENTION

The instant invention is concerned with the preparation of novelcompounds which are prepared by the bioconversion of the known compoundparaherquamide and dihydroparaherquamide in a known microorganism,Cunninghamella blakesleeana MF-4415. Thus, it is an object of thisinvention to describe such novel compounds. It is a further object ofthis invention to describe the process used to prepare such novelcompounds. It is a still further object to describe the novelmicroorganism used to prepare such compounds. Another object is todescribe the use of such compounds as anthelmintic and parasiticidalagents. Additional objects will become apparent from a reading of thefollowing description.

DESCRIPTION OF THE INVENTION

The compounds of the instant invention are best realized in thefollowing structural formulae: ##STR3## wherein A is either a single ora double bond.

In accordance with this invention, novel substances are described whichare prepared by growing under controlled conditions a strain ofmicroorganism identified as Cunninghamella blakesleeana MF-4415, ATCC8688a and including a quantity of paraherquamide ordihyrdoparaherquamide in the fermentation broth. This microorganism isidentified in the Merck Culture Collection as MF-4415 and is publiclyavailable from the American Type Culture Collection at 12301 ParklawnDrive, Rockville, MD 20852 under the accession number ATCC 8688a. Thissame strain has been redeposited at the American Type Culture collectionunder the accession number ATCC 20926.

The instant compounds are produced during the aerobic fermentation ofsuitable aqueous nutrient media under conditions described hereinafter,with a strain of Cunninghamella blakesleeana MF-4415 ATCC 8688a, 20926.Aqueous media such as those used for the production of many antibioticsubstances are suitable for use in the process for the production ofthese compounds.

Such nutrient media contain sources of carbon and nitrogen assimilableby the microorganism and generally low levels of inorganic salts. Inaddition, the fermentation media may contain traces of metals necessaryfor the growth of the microorganisms, and production of the desiredcompound. These are usually present in sufficient concentrations in thecomplex sources of carbon and nitrogen, which may be used as nutrientsources, but can, of course, be added separately to the medium ifdesired.

In general, carbohydrates such as sugars, for example dextrose, sucrose,maltose, lactose, dextran, cerelose, corn meal, oat flour, and the like,and starches are suitable sources of assimilable carbon in the nutrientmedia. The exact quantity of the carbon source which is utilized in themedium will depend, in part, upon the other ingredients in the medium,but it is usually found that an amount of carbohydrate between 1 and 10g/l in the medium is satisfactory. These carbon sources can be usedindividually or several such carbon sources may be combined in the samemedium.

Various nitrogen sources such as yeast hydrolysates, yeast autolysates,yeast cells, tomato paste, corn meal, oat flour, soybean meal, caseinhydrolysates, yeast extracts, corn steep liquors, distillers solubles,cottonseed meal, meat extract and the like, are readily assimilable byCunnighamella blakesleeana MF-4415, ATCC 8688a, 20926 in the productionof the instant compounds. The various sources of nitrogen can be usedalone or in combination in amounts ranging from 1 to 5 g/l in themedium.

Among the nutrient inorganic salts, which can be incorporated in theculture media are the customary salts capable of yielding sodium,potassium, magnesium, ammonium, calcium, phosphate, sulfate, chloride,carbonate, and like ions. Also included are trace metals such as cobalt,manganese, and the like.

It should be noted that the media described hereinbelow and in theExamples are merely illustrative of the wide variety of media, which maybe employed, and are not intended to be limitative.

The following are Examples of media suitable for growing strains ofCunnighamella blakesleeana, MF-4415 ATCC 8688a, 20926:

    ______________________________________                                        Composition of Media                                                          ______________________________________                                        Medium A                                                                      Dextrose                 20     g.                                            Peptone                  5      g.                                            Meat Extract             5      g.                                            Primary Yeast            3      g.                                            NaCl                     5      g.                                            CaCO.sub.3 (after pH adjustment)                                                                       3      g.                                            Distilled Water          1000   ml.                                           pH 7.0                                                                        Medium B                                                                      Tomato Paste             20     g.                                            Modified Starch (CPC)    20     g.                                            Primary Yeast            10     g.                                            CoCl.sub.2 6H.sub.2 O    0.005  g.                                            Distilled Water          1000   ml.                                           pH 7.2-7.4                                                                    Medium C (Slant Medium)                                                       Dextrose                 10.0   g.                                            Bacto Asparagine         0.5    g.                                            K.sub.2 HPO.sub.4        0.5    g.                                            Bacto Agar               15.0   g.                                            Distilled Water          1000   ml.                                           pH 7.0                                                                        Medium D (Seed Medium)                                                        Soluble Starch           10.0   g.                                            Ardamine pH              5.0    g.                                            NZ Amine E               5.0    g.                                            Beef Extract             3.0    g.                                            MgSO.sub.4 7H.sub.2 O    0.5    g.                                            Cerelose                 1.0    g.                                            Na.sub.2 HPO.sub.4       0.190  g.                                            KH.sub.2 PO.sub.4        0.182  g.                                            CaCO.sub.3               0.5    g.                                            Distilled Water          1000   ml.                                           pH 7.0-7.2                                                                    Medium E                                                                      Tomato Paste             40.0   g.                                            Oat Flour                10.0   g.                                            Cerelose                 10.0   g.                                            Corn Steep Liquor        5.0    g.                                            Trace Element Mix        10.0   ml.                                           Distilled Water          1000   ml.                                           pH 6.8                                                                        Trace Element Mix I      1000   ml.                                           FeSO.sub.4.7H.sub.2 O     1000  mg.                                           MnSO.sub.4 .4H.sub.2 O   1000   mg.                                           CuCl.sub.2 .2H.sub.2 O   25.0   g.                                            CaCl.sub.2 .2H.sub.2 O   100.0  mg.                                           H.sub.2 BO.sub.3         56.0   mg.                                           (NH.sub.4).sub.2 MoO.sub.4 .4H.sub.2 O                                                                 10.0   mg.                                           ZnSO.sub.4 .7H.sub.2 O   200.0  mg.                                           Distilled Water          1000   ml.                                           Medium F                                                                      CPC Industrial Starch    40.0   g.                                            Modified (Available from CPC Corp.)                                           Distiller's Solubles     7.0    g.                                            Autolyzed Yeast (Ardamine pH                                                                           5.0    g.                                            available from Yeast Products Inc.)                                           CoCl.sub.2 .6H.sub.2 O   50.0   mg.                                           Distilled Water          1000   ml.                                           pH 7.3                                                                        ______________________________________                                    

The fermentation employing Cunninghamella blakesleeana, MF-4415 ATCC8688 a, 20926 can be conducted at temperatures ranging from about 20° C.to about 40° C. For optimum results, it is most convenient to conductthese fermentations at a temperature in the range of from about 24° C.to about 30° C. Temperatures of about 27°-28° C. are most preferred. ThepH of the nutrient medium suitable for producing the instant compoundscan vary from about 5.0 to 8.5 with a preferred range of from about 6.0to 7.5.

The paraherquamide or dihydroparaherquamide is added to the fermentationof Cunninghamella blakesleeana MF-4415 ATCC 8688 a, 20926 in quantitiesof from 0.1 to 1.0 g per liter of fermentation medium. It is preferredto use from 0.1 to 0.5 g per liter. The paraquamide ordihydroparaherquamide may be added at any time during the fermentationcycle. The compounds may be added to the medium ingredients before theculture is added and the fermentation begins or they may be added duringthe course of the fermentation. In order that the cultures havesufficient time to effect the biotransformation, it is preferred thatthe paraherquamide compounds be added to the fermentation before 50% ofthe cycle is completed, preferably before 25% of the cycle is completed.

Small scale fermentations are conveniently carried out by placingsuitable quantities of nutrient medium in a flask employing knownsterile techniques, inoculating the flask with either spores orvegetative cellular growth of Cunnighamella blakesleeana MF-4415, ATCC8688 a, 20926 loosely stoppering the flask with cotton and permittingthe fermentation to proceed in a constant room temperature of about 28°C. on a rotary shaker at from 95 to 300 rpm for about 2 to 10 days. Forlarger scale work, it is preferable to conduct the fermentation insuitable tanks provided with an agitator and a means of aerating thefermentation medium. The nutrient medium is made up in the tank andafter sterilization is inoculated with a source of vegetative cellulargrowth of Cunnighamella blakesleeana MF-4415 ATCC 8688a, 20926. Thefermentation is allowed to continue for from 1 to 8 days while agitatingand/or aerating the nutrient medium at a temperature in the range offrom about 24° to 37° C. The degree of aeration is dependent uponseveral factors such as the size of the fermentor, agitation speed, andthe like. Generally the larger scale fermentations are agitated at about95 to 300 rpm and about 50 to 500 liters per minute (CFM) of air.

The separation of the novel compound from the whole fermentation brothand the recovery of said compounds is carried out by solvent extractionand application of chromatographic fractionations with variouschromatographic techniques and solvent systems.

The instant compounds have slight solubility in water, but are solublein organic solvents. This property may be conveniently employed torecover the compound from the fermentation broth. Thus, in one recoverymethod, the whole fermentation broth is combined with approximately anequal volume of an organic solvent. While any organic solvent may beemployed, it is preferable to use a water immiscible solvent such asethyl acetate, methylene chloride, chloroform and the like. Generallyseveral extractions are desirable to achieve maximum recovery. Thesolvent removes the instant compound as well as other substances lackingthe antiparasitic activity of the instant compound. If the solvent is awater immiscible one, the layers are separated and the organic solventis evaporated under reduced pressure. The residue is placed onto achromatography column containing preferably, silica gel. The columnretains the desired products and some impurities, but lets many of theimpurities, particularly the nonpolar impurities, pass through. Thecolumn is washed with a moderately polar organic solvent such asmethylene chloride or chloroform to further remove impurities, and isthen washed with a mixture of methylene chloride or chloroform and anorganic solvent of which acetone, methanol, and ethanol and the like arepreferred. The solvent is evaporated and the residue furtherchromatographed using column chromatography, thin layer chromatography,preparative thin layer chromatography, high pressure liquidchromatography and the like, with silica gel, aluminum oxide, ionexchange resins, dextran gels and the like, as the chromatographicmedium, with various solvents and combinations of solvents as theeluent. Thin layer, high pressure, liquid and preparative layerchromatography may be employed to detect the presence of, and to isolatethe instant compounds. The use of the foregoing techniques as well asothers known to those skilled in the art, will afford purifiedcompositions containing the instant compounds. The presence of thedesired compounds is determined by analyzing the various chromatographicfractions for biological activity of physico-chemical characteristics.The structures of the instant compounds have been determined by detailedanalysis of the various spectral characteristics of the compounds, inparticular their nuclear magnetic resonance, mass, ultraviolet andinfrared spectra.

The novel compounds of this invention have significant parasiticidalactivity as anthelmintics, insecticides and acaricides, in human andanimal health and in agriculture.

PREPARATION OF STARTING MATERIALS

Paraherquaride is isolated as a fungal metabolite of Penicilliumparaherquei using standard fermentation and isolation techniques. Theanalytical and structural characteristics of paraherquamide aredescribed in detail in Yamazaki et al Tetrahedron Letters 22 135-136(1981).

Dihydroparaherquamide is prepared from paraherquamide by catalytichydrogenation over palladium on a carbon support. The analyticalcharacteristics of dihydroparaherquamide are also given in Yamazaki etal.

A strain of Penicillium charlesii is identified in the culturecollection of Merck & Co., as MF 5123 and has been deposited at theAmerican Type Culture Collection under the accession number ATCC 20841.The morphological and cultural characteristics of MF 5123 are asfollows:

Morphology of MF 5123

Conidiophores are simple or variously branched with each branch bearinga monoverticillate penicillus and conidial chains forming a long narrowcompact column. Conidiophore walls are smooth or nearly so. Conidia aremostly globose, 1.8μ to 2.4μ, slightly roughened.

Cultural characteristics of MF 5123 Czapek Dox agar

Colonies are white, velvety and raised, becoming grey-green with creamcolored areas. The reverse side is yellowish-brown and cream. As cultureages, aerial growth becomes grayish brown to brown and a tan solublepigment diffuses into medium.

Potato dextrose agar

Colonies are white becoming grey-green, flat, granular. Reverse side isgreenish-brown.

Sabouraud Maltose

Colonies are white becoming brownish green with vectors ofyellowish-tan, raised, velvety. Reverse side is dark brown. A brownsoluble pigment diffuses into agar.

Yeast-extract Malt-extract agar

Colonies are white becoming gray-green with yellowish areas, raised,velvety. Reverse side is dark brown edged with tan.

Corn agar

Colonies are white becoming gray-green, flat, granular. Reverse side isyellowish. As culture ages, it becomes brown with greenish tones.

A comparison with culture descriptions in A Manual of the Penicillia byK. B. Raper and C. Thom and with known cultures show this culture to bea new strain of the known species Penicillium charlesii.

A sample of MF 5123, Penicillium charlesii has been deposited in thepermanent culture collection of the American Type Culture Collection at12301 Parklawn Drive, Rockville, Md. 20852 and has been assigned theaccession number ATCC 20841.

The above description is illustrative of a strain of Penicilliumcharlesii MF 5123 which can be employed in the production of the instantstarting materials. However, the present invention also embraces mutantsof the above described microorganism. For example, those mutants whichare obtained by natural selection or those produced by mutating agentsincluding ionizing radiation such as ultraviolet irradiation, orchemical mutagens such as nitrosoguanidine or the like treatments arealso included within the ambit of this invention.

Paraherquamide may be prepared during the aerobic fermentation of aproducing strain of Penicillium charlesii MF 5123 in either an agitatedaqueous medium or in a static solid medium.

Where the nutrient medium is an aqueous medium, suitable media such asthose used for the production of many antibiotic substances are suitablefor use in this process for the production of paraherquamide.

Such nutrient media contain sources of carbon and nitrogen assimilableby the microrganism and generally low levels of inorganic salts. Inaddition, the fermentation media may contain traces of metals necessaryfor the growth of the microorganisms, and production of the desiredcompound. These are usually present in sufficient concentrations in thecomplex sources of carbon and nitrogen, which may be used as nutrientsources, but can, of course, be added separately to the medium ifdesired.

In general, ingredients such as sugars, for example dextrose, sucrose,maltose, lactose, glycerol, corn, millet, wheat, dextran, cerelose, cornmeal, oat flour, and the like, and starches are suitable sources ofassimilable carbon in the nutrient media. The exact quantity of thecarbon source which is utilized in the medium will depend, in part, uponthe other ingredients in the medium, but it is usually found that anamount between 0.5 and 90% by weight of the medium is satisfactory.These carbon sources may be used individually or several such carbonsources may be combined in the same medium.

Various nitrogen sources such as yeast hydrolysates, yeast autolysates,yeast cells, tomato paste, corn meal, oat flour, soybean meal, caseinhydrolysates, yeast extracts, corn steep liquors, corn, millet, wheat,distillers solubles, cottonseed meal, meat extract and the like, arereadily assimilable by Penicillium charlesii MF 5123 in the productionof the instant compound. The various sources of nitrogen can be usedalone or in combination in amounts ranging from 0.2 to 95% by weight ofthe medium.

Among the nutrient inorganic salts, which can be incorporated in theculture media are the customary salts capable of yielding sodium,potassium, magnesium, ammonium, calcium, phosphate, sulfate, chloride,carbonate, and like ions. Also included are trace metals such as cobalt,manganese, iron, molybdenum, cadmium, zinc, copper, and the like.

It should be noted that the media described hereinbelow and in theExamples are merely illustrative of the wide variety of media, which maybe employed, and are not intended to be limitative.

The following are Examples of media suitable for growing strains ofPenicillium charlesii MF 5123.

    ______________________________________                                        Medium G                                                                      Dextrose                 1.0    g.                                            Soluble starch           10.0   g.                                            Beef extract             3.0    g.                                            Yeast autolysate (As ardamine pH                                                                       5.0    g.                                            available from Yeast Products, Inc.,                                          Clifton, N.J.)                                                                NZ Amine-E (casein hydrolysate-                                                                        5.0    g.                                            available from Humko-Sheffield-                                               Memphis, Tenn.)                                                               MgSO.sub.4 .7H.sub.2 O   0.05   g.                                            Phosphate Buffer         2.0    ml                                            CaCO.sub.3               0.5    g.                                            Distilled water          1000   ml.                                           pH 7.0-7.2                                                                    Phosphate Buffer                                                              KH.sub.2 PO.sub.4        91.0   g                                             Na.sub.2 HPO.sub.4       95.0   g                                             Distilled water          1000   ml                                            pH 7.0                                                                        Medium H                                                                      Tomato paste             20.0   g.                                            Primary yeast            10.0   g.                                            Dextrin (CPC starch)     20.0   g.                                            CoCl.sub.2 .6H.sub.2 O   0.005  g.                                            Distilled water          1000   ml                                            pH 7.2-7.4                                                                    Medium I                                                                      Corn meal                20.0   g.                                            Distillers solubles      10.0   g.                                            Soybean meal             15.0   g.                                            Sodium citrate           4.0    g.                                            CaCl.sub.2 .2H.sub.2 O   0.5    g.                                            MgSO.sub.4 .7H.sub.2 O   0.1    g.                                            CoCl.sub.2 .6H.sub.2 O   0.01   g.                                            FeSO.sub.4 .2H.sub.2 O   0.01   g.                                            Polyglycol P2000 (Polypropylene glycol                                                                 2.5    mg.                                           mw 2000)                                                                      Distilled water          1000   ml.                                           pH 6.5                                                                        Medium J                                                                      Lactose                  20.0   g.                                            Distillers solubles      15.0   g.                                            Autolyzed yeast (Ardamine pH)                                                                          5.0    g.                                            Distilled water q.s. to  1000   ml                                            pH 7.0                                                                        Medium K                                                                      Tomato paste             40.0   g.                                            Oat Flour                10.0   g.                                            Distilled water          1000   ml                                            pH 7.0                                                                        Medium L                                                                      Corn Steep Liquor        15.0   g.                                            (NH.sub.4).sub.2 SO.sub.4                                                                              4.0    g.                                            CaCO.sub.3               6.0    g.                                            Soluble Starch           20.0   g.                                            Corn meal                1.0    g.                                            Soybean meal             4.0    g.                                            Glucose                  5.0    g.                                            KH.sub.2 PO.sub.4        0.3    g.                                            Lard oil                 2.5    g.                                            Distilled water          1000   ml.                                           pH 6.7                                                                        Medium M                                                                      Dextrose                 10.0   g                                             Asparagine               1.0    g                                             K.sub.2 HPO.sub.4        0.1    g                                             MgSO.sub.4 .7H.sub.2 O   0.5    g                                             Yeast Extract            0.5    g                                             Oat Flour                10.0   g                                             CaCO.sub.3               3.0    g                                             Trace Element Mix        10.0   ml                                            Distilled water          1000   ml                                            Adjust pH to 7.2                                                              Trace Element Mix I                                                           FeSO.sub.4 .H.sub.2 O    1000   mg                                            MnSO.sub.4 .4H.sub.2 O   1000   mg                                            CuCl.sub.2 .2H.sub.2 O   25     mg                                            CaCl.sub.2 .2H.sub.2 O   100    mg                                            H.sub.3 BO.sub.3         56     mg                                            (NH.sub.4).sub.6 MO.sub.4 O.sub.24.6H.sub. 2 O                                                         19     mg                                            ZnSO.sub.4 .7H.sub.2 O   200    mg                                            Distilled water          1000   ml                                            Medium N                                                                      Medium G                 1000   ml                                            Oat Flour                10     g                                             pH 7.2                                                                        ______________________________________                                    

The fermentation employing Penicillium charlesii MF 5123 can beconducted at temperatures ranging from about 20° C. to about 40° C. Foroptimum results, it is most convenient to conduct these fermentations ata temperature in the range of from about 20° C. to about 30° C.Temperatures of about 24°-26° C. are most preferred. The pH of thenutrient medium suitable for producing the instant compounds can varyfrom about 3.0 to 8.5 with a preferred range of from about 4.0 to 7.0.

Small scale fermentations are conveniently carried out by placingsuitable quantities of nutrient medium in a flask employing knownsterile techniques, inoculating the flask with either spores orvegetative cellular growth of Penicillium charlesii MF 5123 looselystoppering the flask with cotton and permitting the fermentation toproceed at a constant room temperature of about 25° C. on a rotaryshaker at from 0 to 300 rpm for about 2 to 21 days. For larger scalework, it is preferable to conduct the fermentation in suitable tanksprovided with an agitator and a means of aerating the fermentationmedium. The nutrient medium is made up in the tank and aftersterilization is inoculated with a source of vegetative cellular growthof Penicillium charlesii MF 5123. The fermentation is allowed tocontinue for from 5 to 20 days while agitating and/or aerating thenutrient medium at a temperature in the range of from about 20° to 28°C. The degree of aeration is dependent upon several factors such as thesize of the fermentor, agitation speed, and the like. Generally thelarger scale fermentations are agitated at about 95 to 300 RPM and about2 to 20 cubic feet per minute (CFM) of air.

The fermentation of Penicillium charlesii MF 5123 is also successfullycarried out in a solid fermentation medium under static, that is,non-agitated, conditions. The solid phase aerobic fermentation utilizesthe same sources of carbon, nitrogen and inorganic salts as are used forthe above-described submerged aqueous fermentation with the primarydifferences in the constitution of the medium being the quantity ofwater present. The solid phase fermentations constitute from 30 to 80%by weight of water. Where in comparison with a submerged fermentationmedium which may utilize from 10 to 100 g of solid ingredients per literof medium (1 to 10% w/v), a solid phase medium will contain from 20 to70% w/v of the solid ingredients.

The solid phase fermentation may be carried out aerobically bymaintaining a large ratio of surface area to the mass of the medium.This is readily accomplished by utilizing a 0.3 to 8 cm depth of mediumin a fermentation tray or flask. Since the medium is not mechanicallyagitated, this ensures the presence of sufficient oxygen for growth.Alternatively the solid phase fermentation may be carried out in specialtrays fitted with sterile gauze for passing air through the solid mediumor across the top thereof. Optionally the solid phase fermentation maybe carried out with a tight fitting cover.

The fermentation of large scale portions of media may be carried out instages of increasing quantities of media and it is not necessary thatall of the stages be of the same type, that is aqueous or solid. It hasbeen found to be preferable to carry out the initial stages offermentation in aqueous media and transfer the media to larger scalesolid media.

The compound of this invention is found primarily in the mycelium ontermination of the Penicillium Charlesii MF 5123 fermentation and may beremoved and separated therefrom as described below.

The separation of the Paraherquamide from the whole fermentation brothand the recovery of said compound is carried out by solvent extractionand application of chromatographic fractionations with variouschromatographic techniques and solvent systems.

The instant compound has slight solubility in water, but is soluble inorganic solvents. This property may be conveniently employed to recoverthe compound from the fermentation broth. Thus, in one recovery method,the whole fermentation broth is combined with approximately an equalvolume of an organic solvent. While any organic solvent may be employed,it is preferable to use a water immiscible solvent such as ethylacetate, methylene chloride, chloroform and the like. Generally severalextractions are desirable to achieve maximum recovery. The solventremoves the instant compound as well as other substances lacking theantiparasitic activity of the instant compound. If the solvent is awater immiscible one, the layers are separated and the organic solventis evaporated under reduced pressure. The residue is placed onto achromatography column containing preferably, silica gel. The columnretains the desired products and some impurities, but lets many of theimpurities, particularly the non-polar impurities, pass through. Thecolumn is washed with a moderately polar organic solvent such asmethylene chloride or chloroform to further remove impurities, and isthen washed with a mixture of methylene chloride or chloroform and anorganic solvent of which acetone, methanol, and ethanol and the like arepreferred. The solvent is evaporated and the residue furtherchromatographed using column chromatography, thin layer chromatography,preparative layer chromatography, high pressure liquid chromatographyand the like, with silica gel, aluminum oxide, ion exchange resins,dextrans gels and the like, as the chromatographic medium, with varioussolvents and combinations of solvents as the eluent. Thin layer, highpressure, liquid and preparative layer chromatography may be employed todetect the presence of, and to isolate paraherquamide. The use of theforegoing techniques as well as others known to those skilled in theart, will afford purified compositions containing the instant compound.

The instant compounds are potent endo-and ecto-antiparasitic agentsagainst parasites particularly helminths, ectoparasites, insects, andacarides, infecting man, animals and plants.

The disease or group of diseases described generally as helminthiasis isdue to infection of an animal host with parasitic worms known ashelminths. Helminthiasis is a prevalent and serious economic problem indomesticated animals such as swine, sheep, horses, cattle, goats, dogs,cats and poultry. Among the helminths, the group of worms described asnematodes causes widespread and often times serious infection in variousspecies of animals. The most common genera of nematodes infecting theanimals referred to above are Haemonchus, Trichostrongylus, Ostertagia,Nematodirus, Cooperia, Ascaris, Bunostomum, Oesophagostomum, Chabertia,Trichuris, Strongylus, Trichonema, Dictyocaulus, Capillaria, Heterakis,Toxocara, Ascaridia, Oxyuris, Ancylostoma, Uncinaria, Toxascaris andParascaris. Certain of these, such as Nematodirus, Cooperia, andOesophagostomum attack primarily the intestinal tract while others, suchas Haemonchus and Ostertagia, are more prevalent in the stomach whilestill others such as Dictyocaulus are found in the lungs. Still otherparasites may be located in other tissues and organs of the body such asthe heart and blood vessels, subcutaneous and lymphatic tissue and thelike. The parasitic infections known as helminthiases lead to anemia,malnutrition, weakness, weight loss, severe damage to the walls of theintestinal tract and other tissues and organs and, if left untreated,may result in death of the infected host. The compounds of thisinvention have unexpectedly high activity against these parasites, andin addition are also active against Dirofilaria in dogs,Nematospiroides, Syphacia, Aspiculuris in rodents, arthropodectoparasites of animals and birds such as ticks, mites, lice, fleas,blowfly, in sheep Lucilia sp., biting insects and such migratingdiperous larvae as Hypoderma sp. cattle, Gastrophilus in horses, andCuterebra sp. in rodents.

The instant compounds are also useful against parasites which infecthumans. The most common genera of parasites of the gastro-intestinaltract of man are Ancylostoma, Necator, Ascaris, Strongyloides,Trichinella, Capillaria, Trichuris, and Enterobius. Other medicallyimportant genera of parasites which are found in the blood or othertissues and organs outside the gastrointestinal tract are the filiarialworms such as Wuchereria, Brugia, Onchocerca and Loa, Dracunuculus andextra intestinal stages of the intestinal worms Strongyloides andTrichinella. The compouds are also of value against arthropodsparasitizing man, biting insects and other dipterous pests causingannoyance to man.

The compounds are also active against household pests such as thecockroach, Blatella sp., clothes moth, Tineola sp., carpet beetle,Attagenus sp., and the housefly Musca domestica.

The compounds are also useful against insect pests of stored grains suchas Tribolium sp., Tenebrio sp. and of agricultural plants such asaphids, (Acyrthiosiphon sp.); against migratory orthopterans such aslocusts and immature stages of insects living on plant tissue. Thecompounds are useful as a nematocide for the control of soil nematodesand plant parasites such as Meloidogyne sp. which may be of importancein agriculture.

These compounds may be administered orally in a unit dosage form such asa capsule, bolus or tablet, or as a liquid drench where used as ananthelmintic in mammals. The drench is normally a solution, suspensionor dispersion of the active ingredient usually in water together with asuspending agent such as bentonite and a wetting agent or likeexcipient. Generally, the drenches also contain an antifoaming agent.Drench formulations generally contain from about 0.001 to 0.5% by weightof the active compound. Preferred drench formulations may contain from0.01 to 0.1% by weight. The capsules and boluses comprise the activeingredient admixed with a carrier vehicle such as starch, talc,magnesium stearate, or di-calcium phosphate.

Where it is desired to administer the instant compounds in a dry, solidunit dosage form, capsules, boluses or tablets containing the desiredamount of active compound usually are employed. These dosage forms areprepared by intimately and uniformly mixing the active ingredient withsuitable finely divided diluents, fillers, disintegrating agents, and/orbinders such as starch, lactose, talc, magnesium stearate, vegetablegums and the like. Such unit dosage formulations may be varied widelywith respect to their total weight and content of the antiparasiticagent depending upon factors such as the type of host animal to betreated, the severity and type of infection and the weight of the host.

When the active compound is to be administered via an animal feedstuff,it is intimately dispersed in the feed or used as a top dressing or inthe form of pellets which may then be added to the finished feed oroptionally fed separately. Alternatively, the antiparasitic compounds ofthis invention may be administered to animals parenterally, for example,by intraruminal, intramuscular, intratracheal, or subcutaneous injectionin which the active ingredient is dissolved or dispersed in a liquidcarrier vehicle. For parenteral administration, the active material issuitably admixed with an acceptable vehicle, preferably of the vegetableoil variety such as peanut oil, cotton seed oil and the like. Otherparenteral vehicles such as organic preparation using solketal, glycerolformal, and aqueous parenteral formulations are also used. The activecompound or compounds are dissolved or suspended in the parenteralformulation for administration; such formulations generally contain from0.005 to 5% by weight of the active compound.

Although the antiparasitic agents of this invention find their primaryuse in the treatment and/or prevention of helminthiasis, they are alsouseful in the prevention and treatment of diseases caused by otherparasites, for example, arthropod parasites such as ticks, lice, fleas,mites and other biting insects in domesticated animals and poultry. Theyare also effective in treatment of parasitic diseases that occur inother animals including humans. The optimum amount to be employed forbest results will, of course, depend upon the particular compoundemployed, the species of animal to be treated and the type and severityof parasitic infection or infestation. Generally good results areobtained with our novel compounds by the oral administration of fromabout 0.001 to 10 mg per kg of animal body weight, such total dose beinggiven at one time or in divided doses over a relatively short period oftime such as 1-5 days. With the preferred compounds of the invention,excellent control of such parasites is obtained in animals byadministering from about 0.025 to 0.5 mg per kg of body weight in asingle dose. Repeat treatments are given as required to combatre-infections and are dependent upon the species of parasite and thehusbandry techniques being employed. The techniques for administeringthese materials to animals are known to those skilled in the veterinaryfield.

When the compounds described herein are administered as a component ofthe feed of the animals, or dissolved or suspended in the drinkingwater, compositions are provided in which the active compound orcompounds are intimately dispersed in an inert carrier or diluent. Byinert carrier is meant one that will not react with the antiparasiticagent and one that may be administered safely to animals. Preferably, acarrier for feed administration is one that is, or may be, an ingredientof the animal ration.

Suitable compositions include feed premixes or supplements in which theactive ingredient is present in relatively large amounts and which aresuitable for direct feeding to the animal or for addition to the feedeither directly or after an intermediate dilution or blending step.Typical carriers or diluents suitable for such compositions include, forexample, distillers' dried grains, corn meal, citrus meal, fermentationresidues, ground oyster shells, wheat shorts, molasses solubles, corncob meal, edible bean mill feed, soya grits, crushed limestone and thelike. The active compounds are intimately dispersed throughout thecarrier by methods such as grinding, stirring, milling or tumbling.Compositions containing from about 0.005 to 2.0% weight of the activecompound are particularly suitable as feed premixes. Feed supplements,which are fed directly to the animal, contain from about 0.0002 to 0.3%by weight of the active compounds.

Such supplements are added to the animal feed in an amount to give thefinished feed the concentration of active compound desired for thetreatment and control of parasitic diseases. Although the desiredconcentration of active compound will vary depending upon the factorspreviously mentioned as well as upon the particular compound employed,the compounds of this invention are usually fed at concentrations ofbetween 0.00001 to 0.002% in the feed in order to achieve the desiredantiparasitic result.

In using the compounds of this invention, the individual compounds maybe prepared and used in that form. Alternatively, mixtures of theindividual compounds may be used, or other active compounds not relatedto the compounds of this invention.

The compounds of this invention are also useful in combattingagricultural pests that inflict damage upon crops while they are growingor while in storage. The compounds are applied using known techniques assprays, dusts, emulsions and the like, to the growing or stored crops toeffect protection from such agricultural pests.

The following examples are provided in order that this invention mightbe more fully understood; they are not to be construed as limitative ofthe invention.

EXAMPLE 1

Seed cultures were prepared in medium 1 (20 ml in a 250 ml 3-baffleErlenmeyer flask). The seed flasks were inoculated with spores of C.blakeleeana ATCC 8688a, 20926, MF-4415, and incubated on a rotary shaker(220 rpm) at 27° C. for 24 hours. The transformation flasks (40 mlmedium 3 in 250 ml 3-baffle Erlenmeyer flask) were inoculated with 2 mlof seed culture and incubated at 27° C. on a rotary shaker. Methanol wasused to solubilize paraherquamide for addition. The addition was made at0 hour. The cultures were incubated for 2 days in the presence of 0.05mg/ml paraherquamide. Following incubation, the whole broth wasextracted as described in Example 2.

    ______________________________________                                        Media:                 g/L                                                    ______________________________________                                        Seed Medium 1                                                                 Dextrose               4.0    g                                               Nutrient broth         4.0    g                                               Yeast extract          4.0    g                                               Malt extract           10.0   g                                               1000 ml distilled H2O pH                                                                             7.3                                                    Slant Medium 2                                                                Medium A plus agar     20.0   g/L                                             Transformation Medium 3                                                       Same as Medium 1                                                              ______________________________________                                    

EXAMPLE 2

The whole broth (200 ml) from Example 1 was adjusted to pH 7.5 withdilute sodium hydroxide, then centrifuged and filtered through filterpaper to remove the mycelial cake. The filteted broth was extractedthree times with ethyl acetate (3×200 ml). Ethyl acetate extracts werecombined, dried over sodium sulfate, and concentrated under vaccum to anoily residue. The residue was chromtographed on preparative TLC (MerckSilica gel 60 PF₂₅₄) employing methylenechloride:methanol (90:10). Themetabolites, located by UV lamp (254 nm), were scraped off, eluted with50/50 methylenechloride:methanol and concentrated in vacuo. The residuewas dissolved in acetonitrile and subjected to high performance liquidchromatography (HPLC) purification. HPLC was carried out on WhatmanPartisil 10 ODS-3, 9.4 mm×25 cm column and monitored at 260 nm. Themobile phase consisted of an aqueous buffer (0.01M KH₂ PO₄, 0.02Mtriethylamine, and pH adjusted to 7.0) and acetonitrile (65/35). Eachcompound was collected during repeated injections of the above describedextract. The fractions of UV-absorbing material with similar HPLCretention times were pooled and evaporated to dryness in vacuo. Finally,each compound was dissolved in water and further purified using a C₁₈Sep-Pak (Waters Associates) and methanol-water elution solvent to yieldII (3 mg) with a retention time of 10.8 mintues, III (1 mg) with aretention time of 16.4 mintues, and IV (0.5 mg) with a retention time of21.0 mintues wherein A is a double bond.

EXAMPLE 3

Following the procedures of the Examples 1 and 2 usingdihydroparaherquamide in place of paraherquamide, there is obtainedCompounds II, III and IV wherein A is a single bond.

PREPARATION 1

A 5% portion of an 18X150 MM test tube containing 3.5 g of soil anddried culture MF 5123 (ATCC 20841) was used to inoculate an unbaffledErlenmeyer flask containing 50 ml of Medium 4. After three days ofincubation of 28° C., agitated on a rotary shaker (5 cm throw) at 212rpm, a 2.0 ml aliquot of the growth was aseptically transferred to a 250ml Erlenmeyer flask containing Medium 5. After inoculation, Medium 5 wasincubated 7 days without agitation at 25° C. After 7 days of incubation15 ml of sterile distilled water was added to each flask. Incubation wasthen continued at 25° C. for a further seven days with agitation on arotary shaker at 160 rpm.

PREPARATION 2

A 2.0 ml volume of a frozen vegetative growth in 10% glycerol of theculture MF 5123 (ATCC 20841) was used to inoculate an unbaffledErlenmeyer flask containing 50 ml of Medium 4. After 3 days ofincubation at 28° C., agitated on a rotary shaker (5 cm throw) at 212rpm, a 2.0 ml portion of the growth was aseptically transferred to a 250ml Erlenmeyer flask containing Medium 5. After inoculation, Medium 5 wasincubated 7 days without agitation at 25° C. After 7 days of incubation15 ml of sterile distilled water was added to each flask. Incubation wasthen continued at 25° C. for a further 9 days with agitation on a rotaryshaker at 160 rpm.

PREPARATION 3

A 2.0 ml volume of a frozen vegetative growth in 10% glycerol of theculture MF 5123 (ATCC 20841) was used to inoculate an unbaffledErlenmeyer flask containing 50 ml of Medium 4. After 3 days ofincubation at 28° C., agitated on a rotary shaker (5 cm throw) at 212rpm, a 2.0 ml portion of the growth was aseptically transferred to a 250ml Erlenmeyer flask containing Medium 5. After inoculation, Medium 5 wasincubated for 7 days without agitation at 25° C.

    ______________________________________                                        Medium 4                                                                      Corn Steep Liquor      5.0    g                                               Tomato Paste           40.0   g                                               Oat Flour              10.0   g                                               Glucose                10.0   g                                               Trace Elements Mix     10.0   ml                                              Distilled Water q.s.   1000   ml                                              pH 6.8                                                                        Trace Elements Mix II                                                         FeSO.sub.4 .7H.sub.2 O 1.0    g                                               MnSO.sub.4 .4H.sub.2 O 1.0    g                                               CuCl.sub.2 .2H.sub.2 O 25.0   mg                                              CaCl.sub.2             0.1    g                                               (NH.sub.4).sub.6 MoO.sub.2 .4H.sub.2 O                                                               19.0   mg                                              ZnSO.sub.4 .7H.sub.2 O 0.2    g                                               Distilled Water q.s.   1000   ml                                              Medium 5                                                                      Amount per 250 ml Erlenmeyer flask                                            Corn                   10.0   g                                               Yeast extract          0.5    g                                               Sodium tartrate        0.1    g                                               FeSO.sub.4 .7H.sub.2 O 0.01   g                                               L-cysteine             0.1    g                                               Glycerol               0.5    ml                                              CoCl.sub.2 .6H.sub.2 O 0.002  g                                               Distilled Water        15.0   ml                                              Autoclave         20 minutes, 15 pounds, 121° C.                       then Distilled Water   10.0   ml                                              re-autoclave      20 minutes, 15 pounds, 121° C.                       ______________________________________                                    

PREPARATION 4

The contents of six seven day solid media 250 ML fermentation flasksfrom Preparation 3 were combined and extracted twice with six hundredml. of ethyl acetate. The extracts were combined, dried with sodiumsulfate and concentrated to 60 ml.

A three ml. aliquot of the 60 ml concentrate was further concentrated to0.5 ml. This sample was subjected to preparative thin layerchromatography on an E. Merck Silica-Gel 60 (0.5 mm thickness) plateusing a solvent system of 5:5:0.5 v/v/v hexane:methylenechloride:methanol. After chromatography nine zones were selected by U.V.quenching and iodine staining of a template section of the plate. Theselected areas were scraped and each eluted with 2 ml. of methanol. Theeluted samples were labeled A thru I. One hundred mcl of each sample wassubmitted for C. elegans assay. Sample C Rf=0.16 was active against C.elegans. Sample C was concentrated to dryness and the residue taken upin 400 mcl of methanol and subjected to preparative HPLC chromatographyon a DuPont Zorbax ODS 0.94×25 cm column maintained at 60 degrees C. Thechromatography was carried out using an isocratic system of 65:35 v/vmethanol:water at a flow rate of 4 ml/min. for thirty minutes followedby a linear gradient to 100% methanol over 10 minutes and held at 100%methanol for 35 minutes. The effluent stream was monitored at 226 nm anda setting of 1.28 AUFS using a Laboratory Data Control SpectroMonitor IIdetector equipped with a 1 mm path length cell, and a Spectra-PhysicsSP4100 Computing Integrator. Twenty five fractions were collected.Fraction nine with a retention time of 11.3 minutes was active againstC. elegans. Fractions eight thru twelve were combined and concentratedto dryness. The residue was labeled J and submitted for N.M.R. and massspectral analysis and identified as structure I Paraherquamide. Sample Jcontained 1.8 mg. of paraherquamide based on U.V. analysis usingpublished U.V. data (Tetrahedron Letters Vol. 22 pp 135-136 1980).

PREPARATION 5

The contents of sixteen-seven day solid media 250 ml fermentation flasksfrom Preparation 3 were combined and extracted with three one literportions of ethyl acetate. High pressure liquid chromatography (HPLC)analysis of the three extracts indicated that extracts one and twocontained 430 and 130 mg respectively of paraherquamide. Extract threecontained only 4 mg and was discarded. Extracts one and two werecombined and dried with sdium sulfate. The dried extract wasconcentrated to an oily residue. The residue was taken up in methanol toa volume of 40 ml. This solution was chromatographed on a 200 ml columnof LH-20, previously equilibrated with methanol, using methanol at aflow rate of 3 ml./min collecting a 40 ml fore cut followed by sixty-oneseven ml fractions. HPLC analysis showed that fractions 10 thru 16contained paraherquamide Rt=7.9 minutes. Fractions 10 thru 16 werecombined and concentrated to an oily residue. The residue was taken upin 5:5:0.5 v/v/v hexane:methylene chloride:methanol to a volume of 13.5ml. This solution was chromatographed on a 500 ml column of E. MerckSilica-Gel 60 (0.04 to 0.063 mm particle size) previously equilibratedwith the same solvent system at a flow rate of 10 ml/min. A 200 mlforecut was collected followed by 315 eight ml fractions. Fractions werecombined as follows based on T.L.C. analysis. Fractions 150 to 179; 180to 199; 200 to 239; 240 to 270; and 271 to 315. The combined fractionswere labeled A thru E. Samples C and D were combined, concentrated todryness and labeled F. Sample F was taken up in 2 ml of methanol andfiltered. The filtrate was subjected to preparative HPLC chromatographyusing a Whatman Magnum 20 ODS-3 2.2×25 cm column at room temperatureusing an isocratic solvent system of 68/32 v/v methanol/water at a flowof 10 ml/min. The effluent stream was monitored using a Gilson model 116U.V. detector equipped with a 0.05 mm path length cell and a setting of1.28 AUFS, the detector signal being monitored by a Spectra-PhysicsSP4100 Computing Integrator. Thirty fractions were collected. Fractions16 thru 18 were combined, based on the U.V. recording. The combinedfractions were concentrated to dryness to yield 205.4 mg of pureparaherquamide, structure I.

PREPARATION 6

Solid Phase Fermentation. The inoculant was prepared by placing thecontents of one frozen vial of MF-5123 (ATCC 20841) into 54 ml of medium4 in a 250 ml flask and agitated at 28° C. for 48 hours on a rotaryshaker at 220 rpm. At the completion of the fermentation period 10 ofthe fermentation broth was transferred to 800 ml of medium 4 in a 2liter flask and agitated at 28° C. for 48 hours on a rotary shaker at200 rpm.

The production medium was prepared by dissolving all of the ingredientsof medium 6 except the cracked corn in 3.5 l of distilled water andcombining this solution with 3 kg of cracked corn in a 50×75×5 cm filtertray. The tray was sterilized uncovered for 20 minutes at 121° C. Thecontents of the tray were stirred and another tray of the same dimensionwas placed over the first as a lid and taped tightly closed. The coveredtray was autoclaved for an additional 20 minutes at 121° C. The tray wasremoved from the autoclave and allowed to cool for several hours beforeinoculation. After cooling, the tray was inoculated with 1 liter of theinoculant. The inoculant was distributed uniformly throughout the mediumby mixing with a sterile spatula. The lid was taped securely to the trayand the solid culture fermented at 25° C. for several days withoutagitation.

The above solid phase fermentation was repeated using the same procedurewith the exception that after the uncovered sterilization 2.0 liter ofdistilled water was added to the tray.

    ______________________________________                                        Medium 6 (Solid phase production medium)                                      Material        concentration (g/tray)                                        ______________________________________                                        Cracked corn (Agway)                                                                          3000.0                                                        Yeast extract   150.0                                                         Sodium tartrate  30.0                                                         FeSO.sub.4 · 7H.sub.2 O                                                               3.0                                                          CoCl.sub.2 · 6H.sub.2 O                                                               0.6                                                          L-cysteine       30.0                                                         Glycerol        150.0                                                         pH - No adjustment                                                            ______________________________________                                    

PREPARATION 7

A tray of 3 kg of solid media from Preparation 6 and 20 flaskscontaining 80 gm of solid media (2) each from Preparation 3 wereextracted with ethyl acetate using 2×600 ml of solvent per flask and 2×4liters of solvent per tray. The extracts were combined, labeled A andstored at 5° C. A tray of 3 kg solid media (5) and 20 flasks containing80 gm of solid media per flask were extracted with ethyl acetate and theextracts combined and labeled B.

Samples A and B were combined and concentrated, under reduced pressureat 26° C., to 800 ml. This concentrate was further concentrated, usinghigh vacuum and a 40° C. water bath, to a volume of 300 ml. Threehundred ml of methanol was added to the oily concentrate. Precipitationwas observed and the solution was stored at 5° C. overnight. Theprecipitate was removed by filtration and washed with 200 ml ofmethanol. The filtrate and the methanol wash were combined giving avolume of 800 ml which was labeled C. HPLC analysis of Sample Cindicated the presence of 4.3 gm of paraherquamide. Sample C wasconcentrated under reduced pressure, to remove methanol to a finalvolume of 200 ml. To this concentrate 200 ml of acetonitrile was addedand labeled D. Solution D was extracted with 1×400 ml of hexane followedby 3×300 ml hexane extractions. The acetonitrile layer, 310 ml,contained 3.9 gm of paraherquamide, by HPLC analysis. The acetonitrilesolution was concentrated to dryness and labeled E. The hexane extractswere discarded.

PREPARATION 8

Sample E from Preparation 7 was taken up in 1:1 methylene chloride/ethylacetate to a volume of 180 ml. The solution was chromatographed on a 4liter column of silica gel (Grace), which had previously beenequilibrated with ethyl acetate. The chromatography was carried out withethyl acetate at a flow rate of 200 ml/min collecting five four literfractions followed by 24×500 ml fractions. Fractions 3 thru 9 containing1.95 gms of paraherquamide were combined and labeled F. Fraction two,which was highly colored and contained considerable paraherquamide, wasconcentrated to dryness for rechromatography. The residue, which waslabeled G, was taken up in 1:1 methylene chloride:ethylacetate to avolume of 180 ml and chromatographed as above. Five four liter fractionsfollowed by 24×500 ml fractions were collected. Fractions 4 thru 17containing paraherquamide were combined and labeled H. Samples F and Hwere combined. HPLC analysis of the combined fractions indicated theycontained 4 gms of paraherquamide. The combined sample was concentratedto yield 9.5 grams of solids which were labeled J.

PREPARATION 9

Sample J from Preparation 8 was taken up in 3:1 methanol:methylenechloride to a volume of 110 ml and applied to a 2"×6' Sephadex LH 20column previously equilibrated with methanol. Chromatography was carriedout with methanol at a flow rate of 20 ml/min collecting 20 mlfractions. The fractions were analyzed by T. L. C. and HPLC andparaherquamide was found to be present in fractions 155 thru 186.Fractions 155 thru 164 combined and labeled A. Fractions 165 thru 175combined and labeled B. Fractions 176 thru 186 combined and labeled C.Samples A and B were each concentrated to dryness and their residuetriturated with 20 ml of cold ethyl acetate. The triturates werefiltered and the solids combined and labeled D. The filtrates werecombined and labeled E.

PREPARATION 10

Sample D from Preparation 9 was taken up in 80 ml of 2:1 hexane:acetoneand chromatographed, in two 40 ml portions, on silica gel (Grace)previously equilibrated with 2:1 hexane acetone. Portion one waschromatographed on one liter of silica gel and portion two on 700 ml ofsilica gel. Chromatography in both instances was carried out with 2:1hexane:acetone at a flow rate of 20 ml/min collecting 25 ml fractions.Fractions 153 thru 220 and 143 thru 230 from the respective columnscontained a total of 3.5 gms of paraherquamide.

PREPARATION 11

Paraherquamide (5 mg, 0.01 moles) was dissolved in 2 mL of methanol. 10%palladium-on-carbon (5 mg) was added and hydrogenation carried out under40 psig, 23° C. for 2.5 hours. Reaction was 100% completed after 2.5hours and no side products were observed as determined by HPLC and TLC.The reaction mixture was filtered and the catalyst washed with methanol.The combined methanol solutions containing dihydroparaherquamide weretaken to dryness and purified initially by HPLC on an analytical WhatmanODS-3 column using a 60:40, water:methanol 1% acetic acid and 0.1%triethylamine solvent system delivered at 1.5 mL/min. A 40% aqueousmethanol solvent system was used subsequently for final purification.Identification was based on mass spectral and NMR analyses.

What is claimed is:
 1. Compounds having the formulae: ##STR4## wherein Ais a single or a double bond.
 2. The compound of claim 1 wherein A issingle bond.
 3. The compound of claim 1 wherein A is a double bond. 4.The compound of claim 3 which is Compound II.
 5. The compound of claim 3which is Compound III.
 6. A method for the treatment of parasiticinfections in animals which comprises administering to animals infectedwith parasites an effective amount of a compound of claim
 1. 7. Acomposition useful for the treatment of parasitic infections in animalswhich comprises an inert carrier and an effective amount of a compoundof claim 1.